1. Field of the Invention
The present invention relates a method for treating and inhibiting gastric and duodenal ulcers in a patient.
2. Discussion of the Background
Infection by the gram-negative, spiral, microaerophilic bacterium Helicobacter pylori (H. pylori), formerly known as Campylobacter pylori (C. pylori), is a primary cause of non-autoimmune gastritis, is a factor in peptic ulcer disease and is more common in patients with gastric carcinoma. First isolated by Warren (Lancet (1983) 1:1273) and Marshall (Lancet (1983) 1:1273-5), H. pylori has been isolated in gastric tissue biopsies in patients throughout the world. While the precise mechanism of inflammation is not well understood, H. pylori is found in association with the apical surfaces of gastric mucous-secreting cells.
Due to the site specificity of attachment, it has been suggested that there are specific attachment sites for H. pylori which exist on gastric and duodenal mucous-secreting cells. Numerous studies have been undertaken to attempt to identify the specific binding site of H. pylori.
Evans et al (Infection and Immunity (1988) 56:2896-2906) reported that H. pylori binding to an erythrocyte receptor, as measured by hemagglutination inhibition, is preferentially inhibited by N-acetylneuraminyl-.alpha.(2.fwdarw.3)-Gal .beta.1.fwdarw.4 Glc (herein after NeuAc(2.fwdarw.3)-lactose) as compared with N-acetylneuraminyl-.beta.(2.fwdarw.6)-Gal .beta.1.fwdarw.4 Glc (herein after NeuAc(2.fwdarw.6)-lactose). Sialoproteins which contain the NeuAc(2.fwdarw.3)Gal isomer of NeuAc-lactose, i.e., human erythrocyte glycophorin A, fetuin, and human .alpha..sub.2 -macroglobulin, also inhibited H. pylori binding, but at higher concentrations (mg/ml) than that observed for NeuAc(2.fwdarw.3)-lactose, while no inhibition was observed for the corresponding asialoglycoproteins.
Evans et al ibid, measured the hemagglutination inhibiting ability (HIA) of several compounds containing a NeuAc-lactose structure. Based on the hemagglutination inhibition activity, the researches determined that in order to produce 100% HAI, 1.000 mg/ml of .alpha..sub.2 -Macroglobulin was needed, 0.500 mg/ml of fetuin was needed, 0.250 mg/ml of Glycophorin A was needed and 0.078 mg/ml of bovine NeuAc-lactose was needed. Based on their hemagglutination inhibition studies the researches show fetuin to be about 2 times as effective as .alpha..sub.2 -Macroglobulin but only 0.156 times as effective as bovine NeuAc-lactose which comprises about 80% of NeuAc(2.fwdarw.3)-lactose and 20% of NeuAc(2.fwdarw.6)-lactose.
Evans et al (Infection and Immunity (1989) 57:2272-2278) have also observed that H. pylori binds to monolayers of Y-1 mouse adrenal cells. But, this adherence can be prevented by pretreating the Y-1 cells with neuraminidase and is blocked by fetuin. However, it should be noted that there is no relationship between Y-1 mouse adrenal cells and gastric tissue.
Lingwood et al (Lancet (1989) 2:238-241) have reported the isolation of a gastric glycerolipid material which they observed to behave as a receptor for H. pylori. The material was isolated from red blood cells, and mucosal scrapings of pig stomach and human stomach. The investigators postulated that the material was a sulphated alkylacylglycero-lipid, but the actual structure of this material has not been reported. Subsequent investigations (Lingwood et al., Infection and Immunity (1992) 60:2470-2474) showed that this receptor is phosphatidylethanolamine.
Lingwood et al., Infection and Immunity (1992) 61: 2472-2478 report that Helicobacter pylori specifically recognizes phosphatidylethanolamine, gangliotriaosylceramide and gangliotetraosylceramide and the isolation of an S-adhesin which is believed to be responsible for the lipid-binding specificity of this organism. However, none of the compounds which are reported as specifically recognized by H. pylori, are sialylated oligosaccharides.
Tzovelekis et al (Infection and Immunity (1991) 59:4252-4253) reported binding inhibition of H. pylori to HEp-2 cells by gastric mucin. The investigators observed that purified mucin showed the greatest inhibition of H. pylori binding while asialomucin exhibits somewhat diminished inhibition and periodate-oxidized mucin exhibited the lowest level of binding. On these observations, the researchers concluded that sialic acids are at least partially responsible for the binding interaction between H. pylori and human gastric mucin. However, it should be noted that mucin contains a variety of different saccharide groups and linkages.
Boren et al (Science (1993) 262:1892-1895) have reported that Lewis.sup.b blood group and H type I antigens mediate H. pylori attachment to human gastric mucosa.
Fauchere et al Microbial Pathogenesis, 1990 9 427-439 report that H. pylori adherence can be assessed by microtiter assays and involves a bacterial surface material which co-purifies with urease and is different from the N-acetylneuraminyl-lactose binding hemagglutinin.
Robinson et al report in J. Med. Microbiol. (1990) 33 277-284 that pre-treatment of human erythrocytes with neuraminidase from Arthrobacter ureafaciens and Clostridium perfringens abolished hemagglutination by the soluble, but not the cell-associated hemagglutinin, which suggests that sialic acid is not involved in binding inhibition of H. pylori.
Dunn et al Reviews of Infectious Diseases 1991;13(Suppl 8):(S657-64) report binding inhibition studies by Mean Fluorescence Intensity by treatment of materials with a neuraminidase. The researchers report a 16.8% decrease in MFI upon neuraminidase treatment of N-acetylneuraminyllactose of 16.8%, a 29.8% reduction with fetuin and an 8.6% reduction of asialofetuin. However, the researchers report a 30% increase upon treatment of KATO cells with neuraminidase. Such results call into question the role of sialylation in the site specific binding of H. pylori.
Saitoh et al report a sulfate-containing glycerolipid as a ligand which is specifically recognized by H. pylori.
While there have been numerous studies into compounds with H. pylori binding inhibition, it clear that the literature is replete with conflicting evidence.
Moreover, there is even a lack of a consensus as to the significance of the methods of testing for H. pylori binding inhibition. Hemagglutination assays have been used by many different researchers (see for example Evans et al (Infection and Immunity (1988) 56:2896-2906), however Figueroa et al report in Journal of Infection (1992) 24 263-267, an adherence mechanism, which is not depending on the expression of specific hemagglutinin antigen. This report openly questions the relationship between hemagglutination inhibition and H. pylori binding inhibition. Furthermore, many of the cell surface adhesion systems, used to test for H. pylori binding inhibition, have no relationship to gastric tissue at all.
In addition to the numerous binding inhibition studies, methods have been pursued to treat gastric and duodenal ulcer patients.
Colloidal bismuth subcitrate (CBS) has been used successfully in treating both gastric and duodenal ulcer diseases (for a review, see Lambert in Reviews of Infectious Diseases (1991) 13 (Suppl. 8):S691-5. CBS has proven effective as a histamine H.sub.2 antagonist and has been associated with lower relapse rates after cessation of therapy attributed to CBS's ability to eradicate H. pylori. Bismuth subsalicylate (BSS) has also been observed to inhibit H. pylori.
Coleman et al (U.S. Pat. No. 4,935,406) reported a method for relieving gastrointestinal disorder, resulting from H. pylori population, through the administration of bismuth (phosph/sulf)ated saccharide compositions. The saccharide compositions according to this method are simple phosphates and sulfates of aldose and ketose monosaccharides.
Clinical trials have been reported (Evans et al, Ann. Internal Med. (1991) August 15, 115(4):266-9) in treating H. pylori using ranitidine in conjunction with a "triple therapy" of amoxicillin or tetracycline, metronidazole (an antiprotozoal), and BSS. The clinical studies suggested that ulcer healing was more rapid in patients receiving ranitidine plus the "triple therapy" than in patients receiving ranitidine alone.
The strong role that H. pylori plays in peptic ulcers has led to an announcement in February 1994 by an independent advisory panel of experts convened by the National Institutes of Health, to advise that patients diagnosed with peptic ulcers and H. pylori be treated for two weeks with a combination of antibiotics. A copy of the Consensus Development Conference Statement Helicobacter pylori in Peptic Ulcer Disease is available from the National Institutes of Health. There was no recommendation for any other type of therapy.
However, long-term eradication of this organism has been difficult with these therapies. The antibiotic approach runs the risk of the development of new antibiotic resistant strains. In addition, there are side affects associated from long term antibiotic therapy, which are unpleasant and make compliance with such a treatment regime more difficult. Thus, a method of treating H. pylori with good long-term eradication has not yet been developed.
As evidenced by the prior art identified above, there are a variety of structurally diverse compounds identified as candidates for being responsible for site specific attachment of H. pylori. The state of the art is further complicated by the variety of different in vitro assays used for predicting H. pylori binding inhibition, for which there is no identified correlation with effective H. pylori binding inhibition in mammals (Figueroa et al Journal of Infection (1992) 24 263-267). Even though 3' sialyl lactose has previously been identified as having hemagglutination inhibiting activity, and therefore speculatively identified as being a gastric colonization factor (Evans et al (Infection and Immunity (1988) 56:2896-2906)) it was only one compound of many identified as possible candidates. The same publication, also reports the same activity, albeit only 0.156 times as great, for the compound fetuin. Accordingly, the state of the art, would not allow one to have selected 3' sialyl lactose from the many other and structurally diverse compounds, as a particularly effective means for inhibiting H. pylori binding inhibition in mammals.
Based on the inventors' studies, it has now been discovered that 3' sialyl lactose is a surprisingly effective inhibitor of H. pylori binding inhibition in mammals. And this finding has been validated by the inventors through in vivo mammalian test data.
In addition, contrary to earlier reports, the inventors of the present invention have discovered that fetuin has minimal activity in inhibiting binding of H. pylori cells, in vitro. The inventors have discovered that the binding inhibition activity associated with fetuin, appears to be attributable to a high molecular weight impurity which is a contaminate of commercially available fetuin. Based on their assay, the inventors of the present invention have discovered that 3' sialyl lactose has an ability to inhibit binding of H. pylori to a degree far in excess of what would have been expected in light of that previously reported for fetuin. From previous reports, one would expect that 0.156 times as much 3' sialyl lactose would be needed to achieve the same effectiveness, as achieved with fetuin. But since the inventors of the present invention have discovered that fetuin has minimal effectiveness in binding inhibition of H. pylori cells, their discovery that 3' sialyl lactose surprisingly strongly inhibits H. pylori, provides that 3' sialyl lactose can be used in an amount far below that which would have been predicted from the prior art. It is on the basis of this discovery that the present inventors have realized that 3' sialyl lactose is unexpectedly superior in inhibiting H. pylori in mammals.